Howard Temin: “In from the Cold”

Howard Temin persevered during years of neglect and ridicule until his iconoclastic provirus hypothesis was at last accepted.  This story also features David Baltimore, and personal recollections of Temin and Baltimore.

Concurrently, but independent of each other, Howard Temin and David Baltimore discovered reverse transcriptase in 1970; one of the most dramatic and important findings in the history of molecular biology. The impact of this discovery was so huge that Temin and Baltimore had to wait a mere five years for their contribution to be recognized by the Nobel Committee. Yet the path to the Nobel Prize was far from easy for Temin. Rather, it is a tale of unwavering conviction, persistence, and courage. We begin though with a bit of earlier history.

In 1911, Peyton Rous reported the existence of a filterable, infectious agent that causes sarcomas in chickens. This agent, eventually named the Rous sarcoma virus, was the first virus known to cause solid tumors (1). Moreover; it was the prototype of a virus family that initially was called the RNA tumor viruses (and also the leukoviruses,1).  But, after Temin’s unexpected and radical provirus hypothesis was finally accepted, the family was named the retroviruses.

Here is how Rous described his findings in his original 1911 report: “A transmissible sarcoma of the chicken has been under observation in this laboratory for the past fourteen months, and it has assumed of late a special interest because of its extreme malignancy and a tendency to widespread metastasis. In a careful study of the growth, tests have been made to determine whether it can be transmitted by a filtrate free of the tumor cells . . . . Small quantities of a cell-free filtrate have sufficed to transmit the growth to susceptible fowl.”

Current students might be surprised that Rous’ singularly novel finding of a tumorigenic virus generated only scant interest back in the day. But, bear in mind that nothing whatsoever was known yet regarding the genetic and molecular basis of cancer or, in fact, of the nature of viruses. What’s more, medical researchers in the early 1900s did not recognize the relevance of a transmissible cancer in chickens to malignancies in humans (1).

Indeed, Rous’s unique observations were not appreciated until the 1950s, when it was shown that other viruses (DNA tumor viruses, as well as other RNA tumor viruses) could cause sarcomas, mammary tumors, and leukemias in mice. Only then did the study of tumor viruses become a respectable and even a mainstream pursuit.

Eventually, in 1966, Rous was recognized by the Nobel Committee for his 1911 discovery (2)! This 55-year hiatus is the longest in the history of the Nobel Prize. Bearing in mind that Nobel Prizes are not awarded posthumously, Rous was fortunate to have longevity on his side. He was 87-years-old when awarded the Prize.

This brief anecdote concerning Peyton Rous is relevant to our main account of Howard Temin for multiple reasons. First, the independent discovery of reverse transcriptase by Temin and Baltimore, and the resultant acceptance of Temin’s provirus hypothesis, were the crucial keys to elucidating the unique replication strategy of the retroviruses, of which Rous’ virus was the prototype. Second, reminiscent of the indifferent response of the scientific community to Rous’ discovery, Temin too had to wait years for his contribution to be acknowledged by his peers. However, unique to Temin’s circumstance, during his nearly ten-year wait, he endured being ridiculed by some, while being ignored by most, yet all the time holding fast to his convictions.

Interestingly, during Temin’s 1975 Nobel lecture, he commented as follows on the indifferent response of the scientific community to Rous’ findings at the time they were first reported, and indeed for the next 40 or more years afterwards as well: “Although Rous and his associates carried out many experiments with Rous sarcoma virus, as the virus is now called, and had many prophetic insights into its behavior, they and other biologists of that time did not have the scientific concepts or the technical tools to exploit his discovery.”

Howard M. Temin – via Nobelprize.org. Nobel Media AB 2013

Howard Temin began working on Rous sarcoma virus in the 1950s, while a graduate student in Renato Dulbecco’s laboratory at Caltech [See Renato Dulbecco and the Beginnings of Quantitative Animal Virology]. However, he worked under the direct supervision of Harry Rubin, an early star in the field, who was, at the time, a postdoctoral fellow in the Dulbecco lab. Nothing was known as yet about the replication of the RNA tumor viruses, as the retroviruses were then known. Moreover, little more was known about the molecular basis of cancer in the 1950s than was known in 1911, when Rous first isolated his virus; a state of affairs that would be much alleviated by future studies of the oncogenic retroviruses.

Rubin was a veterinarian by training, perhaps accounting for his somewhat unique appreciation of an oncogenic virus of chickens, well after even Rous himself had lost interest. And, Rubin was responsible for introducing other young investigators to the RNA tumor virus field, both at Caltech and later at UC Berkely.

Rubin’s mentorship of Temin began somewhat fortuitously, as follows. When they first met, Temin was actually doing his graduate research in another laboratory at Caltech, looking into the embryology of the innkeeper worm, Urechis caupo. But he was also serving as a laboratory assistant in the Caltech general biology course. In that capacity, he was dispatched to Dulbecco’s laboratory to obtain some fertilized chicken eggs for use in the general biology lab. Harry Rubin supplied the chicken eggs. But the chance visit from Temin gave Rubin the opportunity to tell Temin about the chicken sarcoma viruses that were being studied in the Dulbecco laboratory.

Rubin had just recently found that he could induce the neoplastic transformation of a normal chicken cell with a single Rous sarcoma virus particle. He then demonstrated that the transformed cell produced hundreds more transformed daughter cells in a week’s time. During their chance conversation, Rubin suggested to Temin that he (Temin) might make use of that observation to develop a quantitative tissue culture assay for Rous sarcoma virus. Sufficiently intrigued by Rubin’s proposition, Temin switched from embryology to virology and proceeded to develop a focus-forming cell culture assay for Rous sarcoma virus; an assay analogous in principle to a plaque assay. But instead of forming plaques of dead cells, the non-cytocidal Rous sarcoma virus induces the growth of visible foci of morphologically transformed neoplastic cells.

Temin’s assay is truly quantitative, since the number of foci of transformed cells that appear in a Rous sarcoma virus-infected cell culture is proportional to the concentration of virus particles in the inoculum. Indeed, Temin’s assay was the first quantitative assay for viral transformation in general, and the first quantitative assay for a retrovirus. Its development was all the more impressive at the time because cell culture was still in its infancy. Importantly, Temin’s assay opened up the study of retroviruses in cell culture, enabling others to eventually make the connection between viral carcinogenesis and the genetic basis of cancer. [Most importantly, the oncogenes of the RNA tumor viruses have their counterparts in the normal cell genome.] But Temin’s more far reaching contribution was still to come. Its genesis was as follows.

As noted above, Rous sarcoma virus is not cytocidal. Consequently, it is able to stably transform chicken cells, while at the same time replicating in them. From Temin’s point of view in the late 1950s, these facts were consistent with the attractive but still untested premise that virus-mediated transformation might result from the continuous presence and expression of virus genes in the transformed cells (3). But how then might one account for the fact that Rous sarcoma virus could likewise stably transform rat cells, which do not support replication of the avian Rous sarcoma virus? To do so, one must either abandon the paradigm that continued viral gene expression maintains the transformed cell phenotype, or one must somehow explain how a viral RNA genome might persist in a line of cells that do not support replication of the virus.

At this early time in the history of retrovirology, it was discovered that bacteriophage λ (lambda) could stably integrate its DNA genome into the chromosome of its host bacterium; a state in which the temperate bacteriophage genome might stably persist for many cell generations. So, did the example of the temperate bacteriophages influence the radical hypothesis that Temin was about to put forward? That question is considered below. In any case, Temin proposed that the Rous sarcoma virus genome likewise persists in eukaryotic host cells in a similar integrated state.

Temin of course realized that such an option would not be possible unless the Rous sarcoma virus single-stranded RNA genome might somehow be transcribed into a double-stranded DNA form. Accordingly, that is precisely what Temin hypothesized. In 1964, Temin’s premise became known as the provirus hypothesis, which explicitly proposes that Rous sarcoma virus generates a DNA copy of its RNA genome, which is then integrated into the cellular DNA as a provirus and stably maintained in that state in clones of transformed cells.

Temin’s provirus hypothesis was iconoclastic in the extreme, largely because it was proposed at the time that the “central dogma of molecular biology” was taking hold. The central dogma, as expounded by Francis Crick in 1958, maintained that information in biological systems always “flows” from DNA to RNA and then to protein. Indeed, the central dogma had taken such a strong hold on thinking at the time that Temin’s provirus hypothesis was regarded as the scientific equivalent of heresy. Consequently, Temin had to fight a long, lonely battle against the criticism and ridicule that his hypothesis generated. Even Harry Rubin, who recruited Temin to the RNA tumor virus field and who served as his first mentor, was candid in his disdain for the provirus hypothesis.

It may now seem odd that the central dogma should have exercised such a strong influence, since RNA viruses were already known and appeared to be an exception to the dogma as then stated. In this regard, Temin noted the following in his 1975 Nobel lecture. “Studies with the newly discovered RNA bacteriophage [see The Phage in the Letter] and with animal RNA viruses, especially using the antibiotic actinomycin D, indicated that RNA viruses transferred their information from RNA to RNA and from RNA to protein and that DNA was not directly involved in the replication of these RNA viruses.”

Temin was a serious researcher, and he indeed carried out experiments to test his radical hypothesis during the ten years that he was being ignored or else scoffed at. Moreover, his experimental results were entirely consistent with his proposal. However, the experimental results generated by the technology of the day could not provide the compelling proof that was demanded to verify a hypothesis that seemingly challenged such strongly entrenched beliefs as enunciated by the central dogma.

Temin’s early experimental findings included the following. First, he reported that actinomycin D, which inhibits transcription from a DNA template, impaired replication of Rous sarcoma virus in cultured chicken cells. Yet while this experimental finding is consistent with the provirus hypothesis, the result also might be explained if viral replication were dependent on the expression of particular cellular genes. Second, Temin showed that inhibitors of DNA synthesis impeded Rous sarcoma virus replication. Yet one could argue that cellular DNA synthesis rather than viral DNA synthesis is necessary for virus replication, perhaps as part and parcel of establishing an intracellular milieu conducive to viral replication. Third, Temin carried out nucleic acid hybridization experiments to demonstrate the presence of Rous sarcoma virus DNA in virus-transformed rat cells. Results of those hybridization experiments also supported the provirus hypothesis. Nevertheless, the hybridization technology of the 1960s could not generate data that was compelling enough to convince Temin’s critics.

In Temin’s Nobel lecture, he said the following about how his experimental findings were received by his colleagues: “Based on the results of these experiments, I proposed the DNA provirus hypothesis at a meeting in the Spring of 1964—the RNA of infecting Rous sarcoma virus acts as a template for the synthesis of viral DNA, the provirus, which acts as a template for the synthesis of progeny Rous sarcoma virus RNA. . . . At this meeting and for the next 6 years this hypothesis was essentially ignored.”

A crucial sticking point preventing acceptance of the provirus hypothesis was that no enzyme was known that could copy RNA into DNA. Thus, the breakthrough that led to widespread acceptance of Temin’s hypothesis came in 1970, when Temin (at the University of Wisconsin) and David Baltimore (at MIT) concurrently, but independently, showed that the previously unknown enzyme, reverse transcriptase, is present in RNA tumor virus particles.Temin found the enzyme in Rous sarcoma virus, while Baltimore found it in the related murine leukemia virus.

In recognition of their independent discoveries, Temin and Baltimore were awarded Nobel Prizes in Physiology or Medicine in 1975. Renato Dulbecco shared in that award for his work on the DNA tumor viruses [see Renato Dulbecco and the Beginnings of Quantitative Animal Virology]. One of Dulbecco’s findings, relevant in the current context, is that neoplastic transformation by the polyomaviruses is associated with the stable integration of their DNA genomes into the host cell genome.

Temin and Baltimore had somewhat different motivations for carrying out their breakthrough experiments. Temin’s entire research career was dedicated to studying the RNA tumor viruses and his primary goal became to validate his provirus hypothesis.  His impetus to seek a reverse transcriptase activity in the retrovirus particle came about as follows. In 1969, in a study that never was reported in its entirety, Temin and his postdoc, Satoshi Mizutani, did experiments demonstrating that the alleged Rous sarcoma virus DNA synthesis could occur in the absence of de novo protein synthesis. This experimental finding implied (to believers at least) that the DNA polymerase activity that catalyzed the synthesis of the viral DNA was present before infection. With no precedent for such a cellular activity, Temin and Mizutani sought the putative polymerase in the virus particle.

In contrast, Baltimore had been studying polymerases generated by other RNA viruses, specifically, ones that transcribe RNA from RNA. Earlier, with his wife, Alice Huang, and other colleagues, he sought to explain why the purified single-stranded RNA genome of vesicular stomatitis virus (VSV) is not infectious, while the purified single-stranded RNA genome of poliovirus is. The contrasting infectivities of the VSV and poliovirus RNA genomes led Baltimore, Huang, and their coworkers to hypothesize that VSV has an obligate requirement for a particle-associated polymerase activity. Earlier, they found that during infection, the poliovirus genome functions as an mRNA after it is released from the virus particle into the cytoplasm. Consequently, the purified poliovirus genome likewise may function as an mRNA in transfection experiments. In contrast to the poliovirus case, they found that the VSV RNA genome is complementary to the mRNAs that encode that virus’s proteins. Accordingly, to account for how VSV might initiate transcription upon infection, they looked for, and found, an RNA-dependent transcriptase activity within VSV particles. From this background and point of view, Baltimore sought a reverse transcriptase activity in retrovirus particles.

David_Baltimore_NIH
David Baltimore in the 1970’s. Image via the National Library of Medicine (image in public domain).

During his Nobel lecture, Baltimore reflected on how his earlier experiences might have led him to his discovery of the retrovirus reverse transcriptase, as follows. “If we look back to virology books of 15 years ago, we find no appreciation yet for the variety of viral genetic systems used by RNA viruses. Since then, the various systems have come into focus, the last to be recognized being that of the retroviruses (“RNA tumor viruses”). As each new genetic system was discovered, it was often the identification of an RNA or a DNA polymerase that could be responsible for the synthesis of virus-specific nucleic acids that gave the most convincing evidence for the existence of the new system. . . .” [This line of thought underlies the Baltimore classification system. See my personal recollection of David Baltimore, below.]

Baltimore spoke after Temin at the Nobel ceremony. The following item from Baltimore’s Nobel lecture is cited here for his comments about his co-award recipient: “In his Nobel lecture, Howard Temin has outlined how he was led to postulate a DNA intermediate in the growth of RNA tumor viruses. Although his logic was persuasive and seems in retrospect to have been flawless, in 1970 there were few advocates and many skeptics. Luckily, I had no experience in the field and so no axe to grind—I also had enormous respect for Howard dating back to my high school days when he had been the guru of the Summer School I attended at the Jackson Laboratory in Maine. So I decided to hedge my bets—I would look for either an RNA or a DNA polymerase in virions of RNA tumor viruses. . . .”

Temin died in 1994 from lung cancer. He was only 59 years of age and never was a smoker. His lung cancer was an adenocarcinoma, a type not linked to smoking. In fact, Temin was a zealous crusader against smoking. Even at the Nobel Prize ceremony in his honor, he reprimanded smokers (including Swedish princesses) in the audience.

After Temin’s death, Baltimore wrote the following: “Ten years in the scientific wilderness is a long time; few have had to bear the silence of their colleagues for so long. I can remember meetings in the 1960s when Howard would present his latest data supporting the provirus notion only to be greeted by either skeptical questions or quiet, polite disbelief. Howard’s conviction that there had to be a provirus never seemed to waver over the whole decade. He knew he was right—and he was—but what fortitude it took to keep looking for the experiment that would show it! My first reaction when I realized that I had seen the reverse transcriptase was to call Howard, because I so much wanted him to know that he was vindicated in his commitment to the idea of a provirus. But he had already found out for himself.”

Howard Temin and David Baltimore published their independently discovered, singularly important finding in back-to-back papers in the British journal Nature (4, 5).

In 1972, Hill and Hillova provided further proof that RNA tumor virus genomes persist in transformed cells in the form of a DNA copy. They did so by demonstrating that purified DNA from a Rous sarcoma virus-transformed cell could produce infectious virus when transfected into a normal cell.

Indisputable evidence of reverse transcription led to the RNA tumor viruses becoming known as the retroviruses. What’s more, it led to the realization that the central dogma, as initially stated by Crick, is not always valid. Exceptions include other viruses such as the hepatitis B viruses, which actually have double-stranded DNA genomes, yet make use of reverse transcription to replicate those genomes. And, there are the cellular retroelements and telomeres, which also bring reverse transcription into play. Importantly, knowledge of the reverse transcription step in the retrovirus life cycle also led to a more complete understanding of HIV infection and the pathogenesis of AIDS. What’s more, the reverse transcriptase enzyme is a critical tool of modern molecular biology.

Interestingly, the discovery of reverse transcriptase led Crick to state that he never excluded the possibility that RNA might serve as a template to make DNA, but instead only the possibility that a protein might serve as the template to make a nucleic acid. Moreover, Crick’s use of the word “dogma” itself caused him additional embarrassment. In his autobiography, What Mad Pursuit, he wrote the following: “I called this idea the central dogma, for two reasons, I suspect. I had already used the obvious word hypothesis in the sequence hypothesis, and in addition I wanted to suggest that this new assumption was more central and more powerful. … As it turned out, the use of the word dogma caused almost more trouble than it was worth. Many years later Jacques Monod pointed out to me that I did not appear to understand the correct use of the word dogma, which is a belief that cannot be doubted. I did apprehend this in a vague sort of way but since I thought that all religious beliefs were without foundation, I used the word the way I myself thought about it, not as most of the world does, and simply applied it to a grand hypothesis that, however plausible, had little direct experimental support.”

We conclude this part of the posting by returning to an issue raised above: did the discovery of bacteriophage lysogeny prompt Temin to put forward the provirus hypothesis? Here are Baltimore’s comments on this question: “Although the pregnant analogy to known lysogenic bacteriophage might have guided Howard, people who were at Caltech at that time assure me that Howard was unlikely to have arrived at the notion of a DNA intermediate through this route. Apparently, the influence of Max Delbrück (6)—who was totally committed to the study of lytic phages and did not really believe in the importance of phage lysogeny—was so great that there was little discussion of lysogeny at Caltech then. Furthermore, Howard has minimized the importance of lysogeny as a precursor to his concepts. Therefore, he must have arrived at the concept of a DNA intermediate simply from the persuasive power of such a concept to explain the properties of the transformed state. He was particularly influenced by the morphological difference between cells transformed by particular Rous sarcoma virus variants, which he felt had to mean that the viral genome continued forever to affect the transformed cell.” Temin, in his Nobel lecture, indeed cites the mutant studies to which Baltimore refers as a key factor in the genesis of the provirus hypothesis.

Personal Recollections of Howard Temin and David Baltimore

I briefly met Howard Temin in 1971, when he visited my postdoctoral advisor, Tom Benjamin. Several years later, when I was a still young assistant professor at UMass, I had the chance to spend more time with Howard when he came to present a seminar in my department. After the seminar, I had the privilege of taking Howard to lunch where, much to my delight, he asked me to tell him about my own research. What struck me during this incident was how attentive and responsive Howard was to all I was telling him. He instantly understood the implications of each experiment I described and his mind always seemed to be one experiment ahead of me. When we were finished, I mentioned this impression to him. His response, delivered in a most unassuming way, was “Lenny, I’ve seen a lot of science. There are only so many experiments one can do, and I’ve seen them all.”

Shortly afterwards, while investigating simian virus (SV40) entry into cells, I became interested in the intracellular signals that SV40 appeared to transmit from the cell surface. At the time, cell signaling was already a very big field with a vast literature. However, very little was known or published about virus-induced signaling. I soon found myself snowed under by the enormity of the new-to-me cell signaling literature. Then, I suddenly realized that I was seeing one or more of the same four basic experimental approaches in each of the papers I was reading.  Recalling my lunch with Howard; there are only so many experiments one can do, and I’d become familiar with all that I would need.

David Baltimore (born in 1938) is currently Professor of Biology at Caltech, where he earlier served as President. Before that, he served as a professor at MIT and as President of Rockefeller University. I never had the pleasure of meeting David Baltimore. Nevertheless, he influenced my career in a major way as follows. I earned my Ph.D. in 1969 in the area of bacterial genetics. Then, I spent an all too brief two years as a post-doc studying transformation by the mouse polyomavirus. Next, it seemed I was all too suddenly an assistant professor at UMass, where I was expected to teach an animal virology course to advanced undergraduates and graduate students.

Here then was my dilemma. I was still an expert bacterial geneticist. But, while I was knowledgeable regarding transformation by the tumor viruses, I was far from being an expert animal virologist. Moreover, I never actually had a virology course. My predicament was compounded further by the fact that the virology textbooks of the day were for the most part descriptive. Thus, I was at a loss as to how I might deliver three 50-minute virology lectures per week to advanced undergraduates and graduate students.

With my back seemingly against the wall, I rather fortuitously came across a 1971 review article by David Baltimore, in which he put forward his Baltimore classification system (7).  In brief, the Baltimore classification system is based on the notion that the nature of a viral genome (e.g., double-stranded DNA, plus-strand RNA, minus-strand RNA, etc.) largely determines its expression strategy. Moreover, there were only six classes, or basic strategies in the original Baltimore scheme, and each of the formal virus families fit into one of the Baltimore classes. Although the Baltimore system is not a formal means for classifying viruses, I immediately recognized its didactic potential. Indeed, I had my answer regarding how to teach virology.

At first, I followed Baltimore’s review article, initially basing my lectures on the references therein, which I updated with more recent articles over the next several years. But, the field was progressing too rapidly to go on in this way, so I had to turn to textbooks. However, none of these books was organized around the Baltimore classification system, which remained the key concept around which I structured my lectures. Indeed, to this day I remain convinced that the best way to teach virology is to discus viruses in the context of virus families, with the Baltimore system as the organizing principle.

Over the years I fantasized about writing a book that might present the field in this way. Virology: Molecular Biology and Pathogenesis was the fulfillment of that vision.

References and notes:

1. An oncogenic virus, later known to be closely related to the Rous sarcoma virus, actually was discovered 3 years earlier by Vilhelm Ellereman and Olaf Bang. They found that leukemia in birds could be transmitted by a filterable agent from leukemic cells or by serum from leukemic birds. However, leukemia was not then recognized as cancer, so the significance of this discovery, like Rous’, went unrecognized.

2. The 1966 Nobel Prize in Physiology or Medicine was divided equally between Peyton Rous “for his discovery of tumour-inducing viruses” and Charles Brenton Huggins “for his discoveries concerning hormonal treatment of prostatic cancer”.

3. Shortly after the discovery of reverse transcriptase, Steve Martin, at the time a graduate student in Harry Rubin’s laboratory (at UC Berkeley where Rubin was then a professor) reported the isolation of a mutant of Rous sarcoma virus that was temperature-sensitive for transformation. Importantly, cells transformed by the mutant virus at its permissive temperature reacquired the normal cell phenotype when incubated at the non-permissive temperature. Thus, expression of a viral oncogene (src in this instance) is required to both initiate and maintain the transformed cell phenotype.

4. Baltimore, D. 1970. RNA-dependent DNA polymerase in virions of RNA tumor viruses. Nature 226:1209-1211.

5. Temin, H. M., S.Mizutani. 1970. RNA-dependent DNA polymerase in virions of Rous sarcoma virus. Nature 226:1211-1213.

6. See the earlier posting, Max Delbruck, Lise Meitner, Niels Bohr,  and the Nazis.

7. Baltimore, D. 1971. Expression of animal virus genomes. Bacteriol Rev. 35:235-41.

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